We examined the biological properties of purified Escherichia coli heat-stable enterotoxin H (STH) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT). The action of STU over time differed from those of STI and CT. STH did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells. Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT. This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT. Subsequently, we examined the involvement of prostaglandins in the action of STlI. The level of prostaglandin E2 in the fluid accumulated as a result of the action of STU increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STH. These results implicate prostaglandin E2 in the mechanism of action of STH.

Escherichia coli heat-stable enterotoxin II (STIU) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STU predicted from the DNA sequence (C. H. Lee, S. L. Moseley, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.Enterotoxigenic Escherichia coli strains produce two kinds of heat-stable enterotoxin (STs), which cause intestinal secretion and diarrhea (1,6,23). One of the proteins is termed STI (also referred to as STa), and the other is STII (also referred to as STb). STI is methanol soluble and active in the suckling mouse assay and the porcine ligated ileum model (2, 23). STI has been well characterized chemically, and the nucleotide sequence of the STI gene has been determined (20)(21)(22). The amino acid residues involved in the expression of STI activity have been determined (4,5,15,19), and a mode of action has been proposed (18).On the other hand, STII is methanol insoluble and active only in the weaned-pig ligated ileum model (2, 7, 24). The gene encoding STII has been cloned and its nucleotide sequence has been determined (10,11,17) (11,17).SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (9), with a gradient polyacrylamide gel (gradient of 4 to 22.5% polyacrylamide). The samples were heated for 10 min at 100°C in the presence of 2% 2-mercaptoethanol and 2% SDS and applied to the gels. Electrophoresis was performed at a constant current of 20 mA for 3 h. The gels were stained with Coomassie brilliant blue and then destained as described in the laboratory manual for the LKB 2117 Multiphor II electrophoresis system (LKB Produkter AB, Bromma, Sweden).Methanol treatment of culture supernatant. E. coli HB101 harboring appropriate plasmids was cultured in 2 ml of Luria broth containing ampicillin (50 ,ug/ml) for pCHL7 and pBR322 or chloramphenicol (50 ,ug/ml) for pWM501 at 37°C overnight with shaking. The supernatant was separated from the cells by centrifugation. A 10-fold excess of analyticalgrade methanol was added to the supernatant. The suspension was allowed to stand at 4°C overnight. The resulting precipitates were collected by centrifugation at 30,000 x g for 1 h. After being washed with methanol, the precipitates were dried under ...

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mSTnuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.

Escherichia coli heat-stable enterotoxin II (STII) is composed of 48 amino acid residues. Among these, one histidine, two arginine, and six lysine residues are basic. Isoelectric focusing showed that the isoelectric point of STII is 9.7, indicating that the side chains of some of these basic amino acid residues project outside the molecule. To understand the role that these basic amino acid residues play in toxicity, STII was chemically modified with ethoxyformic anhydride, maleic anhydride, and phenylglyoxal, which alter the side chains of basic amino acid residues in proteins. Maleic anhydride, which modifies the E amino group, caused a significant loss of enterotoxic activity, but the other two modifiers did not. This indicated that lysine residues play an important role in the expression of the enterotoxic activity of STII and that the contribution of the other basic amino acid residues to the toxicity is relatively low. To confirm this hypothesis, we substituted these nine basic amino acid residues by oligonucleotide-directed site-specific mutagenesis and examined the enterotoxicity of these purified mutant STIls. The enterotoxic activity was reduced when the lysine residues at positions 18, 22, 23, and 46 were substituted. In particular, the substitution at positions 22 and 23 induced a remarkable reduction. These results demonstrate that the lysine residues at positions 22 and 23 are very important in the expression of the enterotoxic activity of STII. Enterotoxigenic Escherichia coli strains produce two kinds of heat-stable enterotoxin (STs), which cause intestinal secretion and diarrhea (2, 10). One is termed STI (also referred to as STa), and the other is STII (also referred to as STb). STI is an 18or 19-amino-acid acidic peptide containing three disulfide

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.